It is now well-recognised that non-competitive sandwich immunoassays generally display higher sensitivity than the more conventional competitive immunoassays. The widely accepted explanation for this higher sensitivity is the use of relatively large amounts both of the immobilised capture binding agent (usually an antibody located on a solid support) and of the labelled developing binding material (also often an antibody). By using large amounts of the antibodies, especially the capture antibody, the rate of reaction between analyte and capture antibody is increased, implying in accordance with the law of mass action that a greater amount of analyte is captured on the solid phase capture antibody in any specified time interval. Thus, the use of large amounts of capture antibody is generally perceived as crucial to the development of non-competitive immunoassays combining very high sensitivity with relatively short incubation times. (See for example Hay et al. "American Thyroid Association Assessment of Current Free Thyroid Hormone and Thyrotropin Measurements and Guidelines for Future Clinical Assays" in Clinical Chemistry, Vol 37, No. 11, (1991) at pages 2002-2008.) This approach nevertheless carries disadvantages. For example, it implies heavy consumption of antibodies which may be scarce and costly to produce. It also involves the use of various stratagems to maximise the total surface area of the solid support on which the capture antibody is deposited. For example, porous glass microspheres have been used as a solid support in sandwich assay systems, the pores greatly increasing the surface area available for antibody attachment.
Roger Ekins has previously argued, for example in WO-84/01031, WO-88/01058 and WO-89/01157, that this general perception is mistaken and that, in certain circumstances, assays which have an even greater sensitivity than that attainable under the conditions mentioned above can be developed when the unknown sample and standard samples containing the analyte are each contacted with such a small amount of the capture binding agent that only an insignificant fraction of the analyte becomes bound to the capture binding agent. (This insignificant fraction is usually less than 5% and ideally 1-2% or less of the total amount of the analyte in the sample, bearing in mind that errors in analyte determination unavoidably introduced into the measuring procedure elsewhere by limitations in the accuracy of sample and reagent manipulation, signal measurements, standardisation, temperature variation and the like are generally of the order of 10% of the analyte in the sample, although sometimes the binding of higher fractions of the analyte up to 10% or so may be tolerated when exact determination is less important.) Only when an insignificant fraction of the total amount of analyte becomes bound is the fractional occupancy F of the binding sites on the capture binding agent related to the concentration [A] of analyte in the sample (at thermodynamic equilibrium) by the equation ##EQU1## where K is the affinity constant of the capture binding agent for the analyte measured at equilibrium and is a constant at a given temperature and other given conditions. Before thermodynamic equilibrium is reached, the above equation also approximately applies (provided that only an insignificant fraction of the analyte in the sample has become bound to the capture binding agent at the time of measurement of the fractional occupancy, irrespective of whether a higher, significant amount becomes bound subsequently, for example by the time equilibrium is reached), subject only to the alteration that in such a situation the constant K in the equation is the apparent affinity constant of the capture binding agent for the analyte at the time of measurement.
It has also been proposed by Roger Ekins in WO-89/01157 etc to carry out such a technique using the capture binding agent spotted onto a solid support in the form of one or more microspots, for example with diameters of 1 mm.sup.2 or less, using sample volumes of the order of 1 ml or less.
However, with such a system a problem may arise to provide a label which can give a sufficiently strong but sensitive signal. Doubts have also been expressed regarding sensitivities attainable using microspot assay formats on the ground that the use of very small amounts of solid-phase capture binding agent must intrinsically necessitate long incubation times and yield low sensitivity assays.